3.9.1. Autophagy, UPR, and Oxidative Stress

In K133AS-R, Atg8 (LinJ.19.0860) that plays an important role in formation of autophagosome was downregulated. Downregulated expression of HSP70 was also observed in K133AS-R parasites. On the other hand, an upregulated expression of lipoate protein ligase (LinJ.36.3230) involved in lipoic acid biosynthesis was observed in K133AS-R parasites, which eventually lead to upregulated expression of γ-glutamyl cysteine synthetase (GSH1).

≤ ≤ **Figure 8.** Susceptibility of K133WT/K133AS-R isolates in the presence of the AQP1 inhibitor or the modulator to ABC transporter, verapamil. In vitro susceptibility of the sensitive wild-type strain K133 WT/artemisinin-resistant strain K133AS-R isolates towards artesunate in the presence of the AQP1 inhibitor (AQP1 Inh.) and verapamil (Vera) at (**A**) the promastigote stage and (**B**) amastigote stage. IC<sup>50</sup> ± SD of three independent experiments in duplicates is represented here. \*\*\* represents *p* ≤ 0.001, \*\*\*\* represents *p* ≤ 0.0001, NS represents not significant, Circle represents IC<sup>50</sup> of K133WT, Triangle represents IC<sup>50</sup> of K133AS-R.

## 3.9.2. Carbohydrate, Lipid, and Amino Acid Metabolism

K133AS-R parasites showed downregulated expression of gene phosphoacetylglucosamine mutase (LmjF07.0805) involved in the conversion of N-acetyl-α-D-glucosamine-1-phosphate to N-acetyl-D-glucosamine-6-phosphate, which later forms fructose-6-phosphate. Further, various glucose transporters, such as the glucose transporter, Imgt2 (LinJ.36.6550, LmjF36.6290), were also downregulated, suggesting downregulation in carbohydrate metabolism. On the other hand, genes involved in amino acid and lipid metabolism, such as methylmalonyl CoA mutase (LinJ.27.0310; involved in isoleucine, valine, and leucine metabolism), glutamine aminotransferases (LinJ.33.1410; involved in glutamine metabolism), myo-inositol-1-phosphate synthase (LinJ.14.1450), and a hypothetical protein having lipase activity (LinJ.13.0200) involved in lipid metabolism, showed upregulated expression.

## 3.9.3. DNA Synthesis and Translation Machinery

Genes responsible for DNA replication like nucleoside transporter 1 (LinJ.36.2040), H1 histone-like protein (LinJ.33.3390), and endonuclease/exonuclease activity (LinJ.28.1000), were downregulated. Genes involved in protein translation, such as translation initiation factor IF-2 (LinJ.33.2880), Isoleucyl-tRNA synthetase (LinJ.36.5870), and 28S ribosomal RNA (LmjF27.rRNA.32), were also downregulated. On the other hand, small RNA molecules that play an essential role in RNA biogenesis and guide chemical modifications of ribosomal RNAs (rRNAs) and other RNA genes (tRNA and snRNAs) U1snRNA, U2 snRNA, and U3 snRNA were upregulated. Further, genes involved in protein degradation, such as metallopeptidase (LinJ.11.0640) and carboxypeptidases (LinJ.33.2670), were downregulated.

**Figure 9.** Transcriptome predicted adaptations contributing artesunate resistance in *L. donovani*: Genes altered in K133AS-R parasites are represented here. Genes marked with an up and down arrow represent, respectively, the upregulated genes and the downregulated genes in K133AS-R parasites. 1, 2, 3, 4, 5, and 6 are probable adaptations in K133AS-R parasites. (1.) Downregulation of Atg8 and HSP70 leads to increased ROS production, which was compensated by upregulation in the expression of GSH1, (2.) Upregulated expression of enzymes involved in amino acid and lipid metabolism and downregulated expression of the enzyme involved in carbohydrate metabolism, suggesting a dependency on these metabolites for energy generation, (3.) Reduced DNA synthesis that leads to the parasites in the quiescence state may be responsible for artesunate resistance in *Leishmania*, (4.) Reduced protein synthesis and reduced protein degradation, (5.) Upregulated expression of AQP1 leads to higher nutrient uptake and increased discharge of waste material and metabolic end product from the parasites and (6.) Upregulated expression of ABC transporter (ABCG1) and partial reversion or resistance in the presence of the ABC transporter modulator verapamil suggested probable involvement of the ABC transporter in the efflux of artesunate drug.
